Methods of treating metabolic disorders

ABSTRACT

A method of increasing the insulin sensitivity of insulin resistant cells includes administering to the cells an amount of all-trans-retinoic acid effective to activate transcription factor perosixome proliferator-activated receptor (PPAR) β/δ of the cells.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.60/938,578, filed May 17, 2007, the subject matter, which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant Nos.NIH-CA068150 and NIH-DK060684 awarded by The National Institutes ofHealth. The United States government has certain rights in theinvention.

TECHNICAL FIELD

The present invention generally relates to a method of treatingmetabolic disorders and more particularly to a method of treatingmetabolic disorders by administering to insulin resistant cellsall-trans-retinoic acid.

BACKGROUND OF THE INVENTION

Metabolic syndrome, also known as syndrome X, is now a global healthproblem of epidemic proportions. This syndrome denotes a collection ofobesity-associated pathologies that include insulin resistance,hyperinsulinemia, enhanced hepatic glucose uptake into the skeletalmuscle and fat, elevated levels of circulating free fatty acids, andincreased fat accumulation in insulin target tissues. The resultinghyperglycemia, dyslipidemia and hypertension also lead to endothelialdysfunction and thus place metabolic syndrome patients at high risk foratherosclerosis. There is an urgent need for elucidation of themolecular events that result in the development of the metabolicsyndrome, and for the identifying novel strategies for prevention andtherapy of the disease.

The molecular events that result in the development of the insulinresistance that underlie the metabolic syndrome remain incompletelyunderstood, but available information suggests that the nuclear hormonereceptors termed peroxisome proliferator activated receptors (PPARs)play central roles in the process. PPARs are ligand activatedtranscription factors that appear to function as “lipid-sensors”. Likeother members of subclass 1 of the superfamily of nuclear hormonereceptors, PPAR s interact with the retinoid X receptor (RXR) to formheterodimers that bind to PPAR response elements in regulatory regionsof specific target genes. Binding of cognate ligands to theseheterodimers result in receptor activation and in upregulation oftranscription of the target genes. PPARs thus induce metabolic cascadesthat upregulate lipid storage, transport, and homeostasis. Three PPARsubtypes, encoded for by three separate genes, are known to exist:PPARα, PPARδ, and PPARγPPARα is expressed in liver, heart, muscle andkidney, where it regulates fatty acid catabolism. PPARγ is expressedpredominantly in adipose tissue and macrophages, where it is involved inadipocyte differentiation, regulation of sugar and lipid homeostasis,and control of inflammatory responses. Thiazolidinediones, syntheticcompounds that activate PPARγ are in current use as antidiabetic drugs.

SUMMARY OF THE INVENTION

The present invention relates to a method of increasing the insulinsensitivity of insulin resistant cells. The method includesadministering to the insulin resistant cells an amount ofall-trans-retinoic acid effective to activate transcription factorperosixome proliferator-activated receptor (PPAR) β/δ of the cells.

In an aspect of the invention, the all-trans-retinoic acid can beadministered at an amount effective to induce expression in the cells ofat least one of 3-phosphoinositide-dependent protein kinase 1 (PDK-1),fasting induced adipose factor (FIAF) or adipose differentiation-relatedprotein (ADRP). The insulin resistant cells can comprise insulinresistant adipocytes, such as insulin adipocytes cells of an obesesubject and/or a subject with metabolic syndrome.

The present invention also relates to a method of treating metabolicsyndrome in a mammalian subject. The method includes administering tothe subject a pharmaceutical composition comprising all-trans-retinoicacid. The subject can include insulin resistant cells, and thepharmaceutical composition can be administered to the subject in anamount effective to increase the insulin sensitivity of the insulinresistant cells.

In an aspect of the invention, the pharmaceutical composition can beadministered at an amount effective to activate transcription factorPPAR β/δ of the insulin resistant cells. The pharmaceutical compositioncan also be administered at an amount effective to induce expression inthe cells of at least one of PDK-1, FIAF, or ADRP. The insulin resistantcells can comprise insulin resistant adipocytes.

The present invention further relates to a method of treating type 2diabetes caused by insulin resistance of cells in a subject. The methodincludes administering to the subject an amount of all-trans-retinoicacid effective to increase the insulin sensitivity of the insulinresistant cells. The all-trans-retinoic acid can be administered at anamount effective to activate transcription factor PPAR β/δ of theinsulin resistant cells. The all-trans-retinoic acid can also beadministered at amount effective to induce expression of at least one ofPDK1, FIAF, or ADRP of the insulin resistant cells.

The present invention still further relates to a method of treatingobesity or an obesity-related condition in a subject. The methodincludes administering to the subject an amount of all-trans-retinoicacid effective to increase the insulin sensitivity of insulin resistantcells in the subject. The all-trans-retinoic acid can be administered atan amount effective to activate transcription factor PPAR β/δ of theinsulin resistant cells. The pharmaceutical composition can also beadministered at amount effective to induce expression in the cells of atleast one of PDK-1, FIAF, or ADRP.

In an aspect of the invention the obesity-related condition can beselected from the group consisting of diabetes 2, metabolic syndrome,hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impairedfasting glucose, dyslipidemia, hypertriglyceridemia, insulin resistance,hypercholesterolemia, atherosclerosis, coronary artery disease,peripheral vascular disease, hypertension, and hepatic steatosis.

The present invention still further relates to a method of treatinghepatic steatosis in a subject. The method includes administering to thesubject an amount of all-trans-retinoic acid effective to reduce hepaticlipid accumulation in the liver. The all-trans-retinoic acid can beadministered at an amount effective to activate transcription factorPPAR β/δ of the liver cells. The pharmaceutical composition can also beadministered at amount effective to induce expression in the cells of atleast one of PDK-1, FIAF, or ADRP.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the present invention will becomeapparent to those skilled in the art to which the present inventionrelates upon reading the following description with reference to theaccompanying drawings, in which:

FIG. 1 illustrates: (A) a graph showing the results of the totalactivation of cells treated with vehicle or GW0742 (GW) or TTNPB (T, 1μM); (B) a graph showing the results of the total activation of cellscotransfected with either control siRNA or siRNA for PPARβ/δ, and thentreated with RA at the denoted concentrations; (C) graphs showing thelevels of mRNA of the PPARβ/δ target genes FIAF, ADRP, and PDK-1expressed from HaCaT cells treated with the denoted ligands (0.1 μM, 4hr); (D) a graph showing the level of ADRP mRNA in HaCaT cells that weretransfected with control siRNA or PPARβ/δ siRNA (24 hr.), and thentreated with the denoted ligands (0.1 μM, 4 hr); (E) immunoblots ofThr-307-phospho-Akt, total Akt, and β-tubulin in cells treated withdenoted ligands (0.1 μM, 12 hr); (F) a graph of the total activation ofHaCaT cells transfected with a RARE-driven luciferase reporter; and (G)a graph of the level of expression of mRNA of the RAR target gene Cyp26ain cells that were treated with the denoted ligands.

FIG. 2 illustrates: (A) titration curves of fluorescence of FABP5titrated with the fluorescence probe ANS; (B) images of COS-7 cellstransfected with an expression vector harboring GFP-FABP5; (C) animmunoblot of HaCaT cells treated with denoted RA or with stearic acid(1 μM, 30 min.); (D) graphs of the results of transactivation assayscarried out in COS-7 cells cotransfected with a luciferase reporterdriven by a PPRE and an expression vector for PPARβ/δ (left panel) orwith an RARE-driven reporter together with an expression vector for RARα(right panel); and (E) a graph of the results of HaCaT cells nottransfected, or transfected with either control siRNA or a constructharboring FABP5 siRNA (24 hr.).

FIG. 3 illustrates immunoblots showing PPAR β/δ associates with the PPREof the ADRP and FIAF genes in liveHaCaT cells.

FIG. 4 illustrates a graph that shows retinoic acid activates PPAR β/δin adipocytes.

FIG. 5 illustrates Whole body weight of mice fed a high fat/high caloriediet in the absence and presence of systemic treatment with RA (0.16mg/day).

FIG. 6 illustrates RA treatment reduces the mass of white adipose tissue(WAT) without affecting the weights of liver, skeletal muscle or brownadipose tissue (BAT).

FIG. 7 illustrates RA treatment increases the food consumption of micemaintained on a high fat diet.

FIG. 8 illustrates RA treatment restores the ability of mice fed a highfat diet to rapidly clear glucose from blood. Control—obese mice fedwith a high fat diet; +RA—mice fed with a high fat diet and treated withRA; lean—mice fed a standard diet.

FIG. 9 illustrates livers of obese mice display pronounced steaosis andRA treatment reverses this detrimental phenotype.

DETAILED DESCRIPTION

It should be understood that the present invention is not limited toparticular methods, reagents, compounds, compositions or biologicalsystems, which can, of course, vary. It should also to be understoodthat the terminology used herein is for the purpose of describingparticular aspects of the present invention only, and is not intended tobe limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the present invention pertains.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred materials and methods are described herein. In describingand claiming the present invention, the following terminology will beused.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of +20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

The term “therapeutically effective amount” refers to an amount ofall-trans-retinoic acid or a derivative thereof that is sufficient toactivate the transcription factor peroxisome proliferator-activatedreceptor δ.

The term “metabolic disorders” refers to a group of identified disordersin which errors of metabolism, imbalances in metabolism, or sub-optimalmetabolism occur. The metabolic disorders as described herein alsoinclude diseases that can be treated through the modulation ofmetabolism, although the disease itself may or may not be caused by aspecific metabolic defect. Such metabolic disorders may involve, forexample, glucose oxidation pathways.

The term “obesity” as used herein is defined in the WHO classificationsof weight. Underweight is less than 18.5 BMI (thin); healthy is18.5-24.9 BMI (normal); grade 1 overweight is 25.0-29.9 BMI(overweight); grade 2 overweight is 30.0-39.0 BMI (obesity); grade 3overweight is greater than or equal to 40.0 BMI. BMI is body mass index(morbid obesity) and is kg/m². Waist circumference can also be used toindicate a risk of metabolic complications. Waist circumference can bemeasured (in cm) at midpoint between the lower border of ribs and theupper border of the pelvis. Other measures of obesity include, but arenot limited to, skinfold thickness and bioimpedance, which is based onthe principle that lean mass conducts current better than fat massbecause it is primarily an electrolyte solution.

The term “obesity-related condition” refers to any disease or conditionthat is caused by or associated with (e.g., by biochemical or molecularassociation) obesity or that is caused by or associated with weight gainand/or related biological processes that precede clinical obesity.Examples of obesity-related conditions include, but are not limited to,type 2 diabetes, metabolic syndrome (i.e., Syndrome X), hyperglycemia,hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose,dyslipidemia, hypertriglyceridemia, insulin resistance,hypercholesterolemia, atherosclerosis, coronary artery disease,peripheral vascular disease, hypertension, and hepatic steatosis.

The term “subject” refers to a mammal, such as a human being. As alsoused herein, the term “subject” may refer to a patient.

The term “pharmaceutical composition” refers to a preparation of one ormore of the agents described herein with other chemical components suchas physiologically suitable carriers and excipients. The purpose of apharmaceutical composition is to facilitate administration of an agentto a subject.

The term “insulin resistance” refers to the condition in which normalamounts of insulin are inadequate to produce a normal insulin responsefrom fat, muscle and liver cells. Insulin resistance in fat cellsresults in hydrolysis of stored triglycerides, which elevates free fattyacids in the blood plasma. Insulin resistance in muscle reduces glucoseuptake whereas insulin resistance in liver reduces glucose storage, withboth effects serving to elevate blood glucose. High plasma levels ofinsulin and glucose due to insulin resistance often leads to metabolicsyndrome and type 2 diabetes.

The term “metabolic syndrome” refers a combination of medical disordersthat increase one's risk for cardiovascular disease and diabetes. It isknown under various other names, such as (metabolic) syndrome X, insulinresistance syndrome, Reaven's syndrome. Symptoms and features arefasting hyperglycemia, diabetes mellitus type 2 or impaired fastingglucose, impaired glucose tolerance, or insulin resistance; high bloodpressure; central obesity (also known as visceral, male-pattern orapple-shaped adiposity), overweight with fat deposits mainly around thewaist; decreased HDL cholesterol; elevated triglycerides; and elevateduric acid levels. Associated diseases and signs are: fatty liver(especially in concurrent obesity), progressing to non-alcoholic fattyliver disease, polycystic ovarian syndrome, hemochromatosis (ironoverload); and acanthosis nigricans (a skin condition featuring darkpatches).

The present invention generally relates to a method of treatingmetabolic disorder associated with insulin resistance of cells or tissueof a mammalian subject. The present invention is based on the discoverythat all-trans-retinoic acid can activate transcription factorperosixome proliferator-activated receptor (PPAR) β/δ in cells of thesubject in which fatty acid binding protein 5 (FABP5) is expressed.Activation of PPAR β/δ in the cells has been shown to induce expressionof at least one of 3-phosphoinositide-dependent protein kinase 1(PDK-1), fasting induced adipose factor (FIAF) or adiposedifferentiation-related protein (ADRP) in the cells. Expression ofPDK-1, FIAF, and ADRP plays a central role in mediating cellresponsiveness to insulin and enabling glucose uptake of the cells aswell as mediating lipid accumulation in the liver.

Based on these discoveries, the present invention provides methods fortreating metabolic disorders associated with insulin resistance, such asobesity, type 2 diabetes, metabolic syndrome, and other obesity relatedconditions.

One aspect of the invention therefore relates to a method increasinginsulin sensitivity in insulin resistant cells of a subject beingtreated. The method includes administering to the insulin resistantcells an amount of all-trans-retinoic acid effective to activatetranscription factor perosixome proliferator-activated receptor (PPAR)β/δ of the cells.

The all-trans-retinoic acid administered to subject has the followinggeneral formula:

The all-trans-retinoic acid is commercially available from, for example,Sigma Chemical (St. Louis, Mo.) and BASF Pharma Solutions. Theall-trans-retinoic acid can also be synthesized as described, forexample, in U.S. Pat. No. 5,808,120, which is herein incorporated byreference in its entirety.

The all-trans-retinoic acid can be administered to the subject in anamount effective to induce expression of at least one of3-phosphoinositide-dependent protein kinase 1 (PDK-1), fasting inducedadipose factor (FIAF) or adipose differentiation-related protein (ADRP)in the insulin resistant cells. In one aspect of the invention, theinsulin resistant cells can comprise insulin resistant adipocytes of anobese subject, a subject with a metabolic disorder, and/or a subjectwith metaboic syndrome. All-trans-retinoic acid administered toadipocytes of the subject can activate PPAR δ in adipocytes, therebyinducing expression of at least one of PDK1, FIAF, or ADRP. Upregulatedexpression of PDK-1 can sensitizes the insulin resistant cell to insulinactivity enabling glucose activity and the treatment of variousmetabolic disorders. Upregulated expression of FIAF and/or ADRP canlower lipid accumulation and fat accumulation in the subject.

The various metabolic disorders associated with insulin resistance thatcan be treated with the all-trans-retinoic acid include, for example,obesity and obesity related conditions (e.g., type 2 diabetes,atherosclerosis), and metabolic syndrome. It will be appreciated thatother metabolic disorders associated with insulin resistance can betreated with the all-trans-retinoic acid in accordance with the presentinvention.

The all-trans-retinoic may be administered to a subject by anyconventional route of administration, including, but not limited to,intravenous, oral, subcutaneous, intramuscular, intradermal andparenteral administration. Preferably, formulations are for parenteral(e.g., intravenous) or oral administration.

The all-trans-retinoic acid can be provided in a pharmaceuticalcomposition for administration to the subject. The dosage of thecomposition including all-trans-retinoic acid administered to thesubject may be varied over a wide range from 1 to 1000 mg per adulthuman per day. For oral administration, the compositions are preferablyprovided in the form of tablets containing, for example, about 0.01,0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200,250 or 500 milligrams of the all-trans-retinoic acid for the symptomaticadjustment of the dosage to the patient to be treated. Theall-trans-retinoic acid may be administered, for example, on a regimenof 1 to 2 times per day. The dosages, however, may be varied dependingupon the requirement of the patients, the severity of the conditionbeing treated and the compound being employed. The use of either dailyadministration or post-periodic dosing may be employed.

The compositions can be provided in unit dosage forms such as tablets,pills, capsules, powders, granules, sterile parenteral solutions orsuspensions, metered aerosol or liquid sprays, drops, ampoules,auto-injector devices or suppositories; for oral parenteral, intranasal,sublingual or rectal administration, or for administration by inhalationor insufflation. Alternatively, the composition may be presented in aform suitable for once-weekly or once-monthly administration; forexample, an insoluble salt of the active compound, such as the decanoatesalt, may be adapted to provide a depot preparation for intramuscularinjection.

For preparing solid compositions, such as tablets, theall-trans-retinoic acid can be mixed with a pharmaceutical carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water, toform a solid homogenous preformulation composition.

When referring to these preformulation compositions as homogeneous, itis meant that the active ingredient or ingredients are dispersed evenlythroughout the composition so that the composition may be readilysubdivided into equally effective dosage forms such as tablets, pillsand capsules. This solid preformulation composition is then subdividedinto unit dosage forms of the type described above containing from 0.1to about 500 mg of all-trans-retinoic acid.

The tablets or pills of the composition can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an entericlayer, which serves to resist disintegration in the stomach and permitsthe inner component to pass intact into the duodenum or to be delayed inrelease. A variety of material can be used for such enteric layers orcoatings, such materials including a number of polymeric acids with suchmaterials as shellac, cetyl alcohol and cellulose acetate.

The liquid forms in which the pharmaceutical compositions includingall-trans-retinoic acid may be incorporated for administration orally orby injection include, aqueous solutions, suitably flavoured syrups,aqueous or oil suspensions, and flavoured emulsions with edible oilssuch as cottonseed oil, sesame oil, coconut oil or peanut oil, as wellas elixirs and similar pharmaceutical vehicles. Examples of dispersingor suspending agents for aqueous suspensions, include synthetic andnatural gums such as tragacanth, acacia, alginate, dextran, sodiumcarboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone orgelatin. The liquid forms in suitably flavored suspending or dispersingagents may also include the synthetic and natural gums, for example,tragacanth, acacia, methyl-cellulose and the like. For parenteraladministration, sterile suspensions and solutions are desired. Isotonicpreparations which generally contain suitable preservatives are employedwhen intravenous administration is desired.

Advantageously, pharmaceutical compositions including all-trans-retinoicacid may be administered in a single daily dose, or the total dailydosage may be administered in divided doses of two, three or four timesdaily. Furthermore, pharmaceutical compositions includingall-trans-retinoic acid can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal skinpatches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

For instance, for oral administration in the form of a tablet orcapsule, the all-trans-retinoic acid can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders; lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum and the like.

Optimal dosages to be administered may be readily determined by thoseskilled in the art, and will vary with the particular compound used, thestrength of the preparation, the mode of administration, and theadvancement of the disease condition. In addition, factors associatedwith the particular patient being treated, including patient age,weight, diet and time of administration, will result in the need toadjust dosages.

The all-trans-retinoic pharmaceutical compositions of the presentinvention can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesicles,and multilamellar vesicles. Liposomes can be formed from a variety oflipids, including but not limited to amphipathic lipids such asphosphatidylcholines, sphingomyelins, phosphatidylethanolamines,phophatidylcholines, cardiolipins, phosphatidylserines,phosphatidylglycerols, phosphatidic acids, phosphatidylinositols, diacyltrimethylammonium propanes, diacyl dimethylammonium propanes, andstearylamine, neutral lipids such as triglycerides, and combinationsthereof. They may either contain cholesterol or may be cholesterol-free.

EXAMPLES Example 1 In HaCaT Keratinocytes, RA Activates PPARβ/δ inParallel to Activation of RAR

The ability of RA to activate PPARβ/δ in the human keratinocyte cellline HaCaT was examined. Transactivation assays were conducted in HaCaTcells transfected with a PPRE-driven luciferase reporter. Cells weretreated with the ligands denoted in FIG. 1 for 15 hours, lysed andluciferase activity was measured and corrected for β-galactosidaseactivity. Data was normalized to the basal activity. Data are mean±SEM,n=3

FIG. 1A illustrates a graph showing the results of the total activationof cells treated with vehicle or GW0742 (GW) or TTNPB (T, 1 μM). FIG. 1Billustrates a graph showing the results of the total activation of cellscotransfected with either control siRNA or siRNA for PPARβ/δ, and thentreated with RA at the denoted concentrations. FIG. 1C illustratesgraphs showing the levels of mRNA of the PPARβ/δ target genes FIAF,ADRP, and PDK-1 expressed from HaCaT cells treated with the denotedligands (0.1 μM, 4 hr). Levels of mRNA of the PPARβ/δ target genes FIAF,ADRP, and PDK-1 were analyzed by Q-PCR and normalized to 18s mRNA. FIG.1D illustrate a graph showing the level of ADRP mRNA in HaCaT cells thatwere transfected with control siRNA or PPARβ/δ siRNA (24 hr.), and thentreated with the denoted ligands (0.1 μM, 4 hr). ADRP mRNA was analyzedby QPCR and normalized to 18s mRNA. Data are mean±SEM, n=3. FIG. 1Eillustrates immunoblots of Thr-307-phospho-Akt, total Akt, and β-tubulinin cells treated with denoted ligands (0.1 μM, 12 hr). Data from arepresentative experiment, which was repeated 4 times with similarresults are shown. FIG. 1F illustrates a graph of the total activationof HaCaT cells transfected with a RARE-driven luciferase reporter. Cellswere treated with RA at the denoted concentrations (15 hr), lysed, andluciferase activity measured and corrected for β-galactosidase activity.Data were normalized to the basal activity. Data are mean±SEM, n=3. FIG.1G illustrates a graph of the level of expression of mRNA of the RARtarget gene Cyp26a in cells that were treated with the denoted ligands.Expression of mRNA of the RAR target gene Cyp26a was analyzed by Q-PCRand normalized to 18s mRNA. Data are mean±SEM, n=3.

Transcriptional activation assays showed that the syntheticPPARβ/δ-selective ligand GW0742, but not the RAR-selective ligand4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid (TTNPB), induced transcription of the reporter (FIG. 1A). Theseobservations attest to the expression and functionality of PPARβ/δ inthese cells and demonstrate specificity of reporter response. RA alsoenhanced the expression of the PPRE-driven reporter and did so in a doseresponsive manner (FIG. 1B). The response was markedly suppressed whenthe expression of PPARβ/δ in the cells was decreased by about 80% bysiRNA methodology (FIG. 1B), indicating that the ability of RA to inducereporter expression was indeed mediated by this receptor and not by RAR.

We then set out to examine the ability of RA to induce the expression ofendogenous PPARβ/δ target genes in HaCaT cells. One of these, PDK-1, isa direct PPARβ/δ target in HaCaT cells. Two other genes, fasting inducedadipose factor (FIAF) and adipose differentiation-related protein(ADRP), are targeted by PPARβ/δ in other cells.

FIG. 3 illustrates PPAR β/δ associates with the PPRE of the ADRP andFIAF genes in live HaCaT cells, verifying that both are direct targetsfor this receptor in the context of these cells. Chromatinimmunoprecipitation assays were conducted in HaCaT cells usingpre-immune IgG or antibodies against PPARβ/δ. The regions containing thePPRE of the ADRP and FIAF genes were amplified using appropriateprimers. A 220 bp region 6-kb upstream from the strat site of GAPDH wasused as a negative control.

The PPARβ/δ ligand GW0742 as well as RA upregulated the expression ofmRNA for all three of these endogenous PPARβ/δ target genes (FIG. 1C).The observations that the RAR-ligand TTNPB had little effect on theexpression of these genes further confirm that RAR is not involved inthis activity of RA. As a control, cells were treated with 9-cis-RA(9cRA), a ligand that activates RXR, the obligatory heterodimerizationpartner for both RAR and PPARs. This ligand induced a modest response,which likely emanated from activation of the RXR moiety of theRXR-PPARβ/δ heterodimer. The small magnitude of the response indicatesthat the induction of ADRP and FIAF by RA is not a result of conversionof RA to its 9-cis isomer within the cells. Further support for theconclusion that upregulation of these genes by RA is mediated by PPARβ/δwas provided by the observations that an 80% decrease in the expressionof this receptor significantly hampered the induction of ADRP by bothGW0747 and RA (FIG. 1D).

Notably, one of the PPARβ/δ targets found to be induced by RA, PDK-1, isan important component of the anti-apoptotic activities of this receptorin keratinocytes, where induction of this kinase leads tophosphorylation and activation of the downstream PDK-1-target survivalfactor Akt. The effects of RA or GW0742 on the phosphorylation level ofAkt were thus examined. Treatment with either of these ligands, but notwith TTNPB or 9cRA, significantly increased the phosphorylation level ofAkt (FIG. 1E).

In addition to activating PPARβ/δ, RA also upregulated the expression ofa reporter gene construct driven by an RAR response element (FIG. 1F),and it efficiently upregulated the expression of mRNA for CYP26a, aknown direct RAR target gene (FIG. 1G). Hence, in HaCaT cells, RAtreatment results in parallel activation of both RAR and PPARβ/δ.

FABP5 Translocates Into the Nucleus in Response to RA, and it EnhancesRA Induced, PPARβ/δ-Mediated Transcriptional Activation.

The observations that RA can activate both RAR and PPARβ/δ raise thequestion of the factors that regulate the dual activity of this hormone.The iLBPs CRABP-II and FABP5 mobilize to the nucleus in response toligands that activate RAR and PPARβ/δ, respectively, and they bind totheir cognate receptors to form a complex through which the ligand isdirectly ‘channeled’ to the receptor. Consequently, CRABP-II enhancesthe transcriptional activity of RAR, while FABP5 facilitates theactivation of PPARβ/δ. The observations that RA serves as a ligand forPPAR β/δ thus raise the possibility that RA may be delivered to thisreceptor by FABP5.

FIG. 2A illustrates titration curves of fluorescence of FABP5 titratedwith the fluorescence probe ANS. Titrations curves (left panel, filledsquares) were corrected for linear non-specific fluorescence (solid lineat end of titration curve), and corrected data (filled circles) analyzedto yield a Kd of 57±7.3 nM (mean±SD, n=3). Kds for the association ofFABP5 with RA (middle panel) and with GW0742 (right panel) weredetermined by fluorescence competition titrations. FIG. 2B illustratesimages of COS-7 cells transfected with an expression vector harboringGFP-FABP5. Images were acquired from live cells before and after a 30min. treatment with RA (1 μM). Right panel: quantitation ofnuclear/cytoplsmic partitioning of FABP5 in cells treated with denotedligands. Forty cells of each treatment group were analyzed (mean±SEM).FIG. 2C illustrates an immunoblot of HaCaT cells treated with denoted RAor with stearic acid (1 μM, 30 min.). Nuclei were separated from cytosolby subcellular fractionation (Calbiochem ProtoExtract SubcellularProteome Extraction kit) and analyzed for the presence of FABP5 byimmunoblots. FIG. 2D illustrates graphs of the results ofTransactivation assays carried out in COS-7 cells cotransfected with aluciferase reporter driven by a PPRE and an expression vector forPPARβ/δ (left panel) or with an RARE-driven reporter together with anexpression vector for RARα (right panel). Cells were also transfectedwith an empty vector or with expression vectors for either FABP5 orCRABP-II, treated with RA, lysed, and luciferase activity determined.Data are mean±SEM, n=3. FIG. 2E illustrates a graph of the results ofHaCaT cells not transfected, or transfected with either control siRNA ora construct harboring FABP5 siRNA (24 hr.). The ability of denotedligands to induce ADRP expression was monitored by Q-PCR and normalizedto 18s mRNA. Data are mean±SEM, n=3.

The fluorescence-based binding assay (FIG. 2A) demonstrated that GW0742and RA bind to FABP5 with Kds of 42.3±4.5 nM, and 34.8±6.6 nM,respectively (mean±SD, n=3), in good agreement with binding affinitiesof this protein towards other ligands. The subcellular localization ofFABP5 was then examined. COS-7 cells were transfected with FABP5 fusedto green fluorescence protein (GFP), and confocal fluorescencemicroscopy was used to image GFP-FABP5 in live cells treated withvarious ligands (FIG. 2B). Similarly to the behavior of GFP whentransfected alone, GFP-FABP5 in untreated cells distributed between thecytoplasm and the nucleus, most likely reflecting that over-expressionof the protein leads to leakage into the nucleus even in the absence ofa specific nuclear localization signal (data not shown). Treatment ofcells with stearic acid, a long chain fatty acid that binds FABP5 butdoes not activate it, did not affect the subcellular distribution of theprotein. In contrast, treatment with either GW0742 or RA resulted in adistinct shift of the protein into the nucleus (FIG. 2B). To monitor theeffects of ligands on the localization of endogenous FABP5 in HaCaTcells, cells were treated with vehicle, RA, or stearate, subjected tosubcellular fractionation, and the presence of FABP5 in cytosol and innuclei examined by immunoblots (FIG. 2C). The data demonstrated thatendogenous FABP5 in HaCaT cells is predominantly cytosolic in theabsence of ligand, and that it accumulates in the nucleus in response toRA, but not upon treatment with stearic acid. Hence, like known PPARβ/δ-ligands, RA activates the nuclear localization of FABP5.

The effects of FABP5 on the ability of RA to activate PPAR β/δ wereexamined by transactivation assay using COS-7 cells, which express verylow level of either FABP5 or CRABP-II. Cells were co-transfected with aluciferase reporter construct driven by a PPRE, an expression vector forPPARβ/δ, and a vector harboring cDNA for either FABP5 or CRABP-II. Cellswere then treated with RA, and the expression of the reporter monitored(FIG. 2D, left panel). RA enhanced the expression of the PPRE-drivenreporter in a dose-responsive manner. While expression of CRABP-II didnot affect the activity, FABP5 significantly enhanced RA-induced,PPARβ/δ-mediated transactivation. To investigate the effect of thebinding proteins on RA-induced activation of RAR, cells were transfectedwith a luciferase reporter under the control of an RAR response element(RARE), an expression construct for RARα, and a vector harboring cDNAfor either FABP5 or CRABP-II. In agreement with previous reports,CRABP-II augmented RAinduced transactivation of RAR. On the other hand,FABP5 had little effect on this activity (FIG. 2D, right panel). Cellsin which the receptors were not ectopically expressed displayedqualitatively similar behaviour but the magnitudes of the ligand-inducedresponses were significantly smaller (not shown).

The involvement of FABP5 in RA-induced activation of PPARβ/δ was furtherinvestigated by examining the effect of decreasing the expression levelof this binding protein on the ability of RA to activate the receptor inHaCaT cells. Cells were transfected with FABP5 siRNA, resulting in anabout 80% decrease in the level of the protein, and induction of thePPARβ/δ target gene ADRP was monitored (FIG. 2E). Decreasing theexpression of FABP5 markedly attenuated the ability of both GW0742 andRA to upregulate the expression of the ADRP, further substantiating thatthe presence of FABP5 is necessary for efficient activation of PPARβ/δby its ligands, including RA.

The present work demonstrates that RA serves as a physiological ligandfor PPARP/6 under some but not all circumstances. However, this receptordisplays near ubiquitous tissue expression, raising the question of thenature of the ligand(s) that activate it in tissues that do not supportactivation by RA. The ligand binding pocket of PPARβ/δ is much largerthan the pockets of other nuclear receptors. It may thus accommodatemultiple ligands, and it has been suggested that various long chainfatty acids and eicosanoids may serve as effective PPARβ/δ activators.Whether some of these ligands function as true physiological ligands forthe receptor remains to be clarified, but the present work and thesimilar nature of ligands that bind to FABPs and PPARs raise thepossibility that FABPs other than FABP5 may act to deliver ligands otherthan RA to PPARβ/δ, and thus that they may regulate the functionality ofdistinct ligands in specific tissues.

When enabled, RA signalling through PPARβ/δ has profound functionalconsequences. One consequence, explored here, is that such signallingevokes potent anti-apoptotic activities that overcome thegrowth-inhibitory activities of RAR, and allow cells to survive in theface of powerful apoptotic agents. Hence, RA-dependent maintenance ofskin integrity, proliferation of basal keratinocytes, and survival ofthese cells during wound repair likely stem from a high expression levelof FABP5, enabling RA to activate PPARP/6.

Experimental Procedures

Reagents CRABP-II antibodies were provided by Pierre Chambon (IGMCB,Strasbourg, France). FABP5 antibodies were purchased from BioVender(Candler, N.C.). Antibodies against full-length and cleaved PARP, andtotal and phospho-Akt (Thr308) were from Cell Signaling Technology.Anti-mouse and anti-rabbit immunoglobulin antibodies conjugated tohorseradish peroxidase were from Amersham (Arlington Heights, Ill.) andBioRad (Hercules, Calif.), respectively. Anilinonaphtalene-8-sulphonicacid (ANS), RA, TNFα, and TRAIL were from Sigma Chemical Co. (St. Louis,Mo.).4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicAcid (TTNPB) and GW0742 were purchased from Biomol International(Plymouth Meeting, Pa.) and Toronto Research Diagnostics Inc. (Toronto,ON), respectively.

Proteins. Histidine-tagged CRABP-II and GST-tagged FABP5 were expressedin the E. coli strain BL21. Bacteria were grown overnight at 25° C. andprotein expression was induced with 0.5 mMisopropyl-β-D-thiogalactopyranoside (IPTG) overnight. Bacteria werepelleted and lysed in lysis buffer (20 mM Tris, pH 8.0, 0.5 mM NaCl, 100μM phenylmethylsulfonyl fluoride) containing lysozyme and DNAse I.Mixtures were incubated (30 min., 37° C.), centrifuged, and proteinspurified by affinity chromatography, and dialyzed against lysis buffer.

Cells. COS-7, HaCaT, NaF, and MCF-7 cells were maintained in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% charcoal-treatednewborn bovine calf serum (Cocalico Biologicals Inc., Reamstown, Pa.).COS-7 cells were transfected using Fugene (Roche DiagnosticsCorporation). Other cell lines were transfected using Superfect(Qiagen).

Transactivation assays. Cells were cultured in 24-well plates andco-transfected with (PPRE)₃-luciferase reporter vector (100 ng), avector harboring the appropriate iLBP (in pCDNA 3.1, 200 ng), andpCH110, a β-galactosidase expression plasmid (50 ng). In someexperiments, an expression vector encoding RAR or PPARβ/δ (in pSG5, 50ng) was cotransfected. Transfections were carried out using Fugene(Roche Diagnostics Corporation) according to the protocol of themanufacturer. Twenty-four hours following transfection, medium wasreplaced by DMEM, and ligands were added (RA—in ethanol, otherligands—in dimethylsulfoxide). Following 24 hr of treatment, cells werelysed and lysates assayed for luciferase activity (Luciferase assaysystem, Promega) which was corrected for β-galactosidase activity.Experiments were carried out in triplicates.

Apoptosis was evaluated using the APOPercentage Apoptosis Assay kit(Biocolor Ltd. United Kingdom). 1×10⁶ cells were suspended in 1 mlmedium and dispensed into 96-well microplates. Cells were grownovernight, treated with appropriate ligand (2 hr.) and apoptosis inducedwith TNFα or TRAIL overnight. Medium was replaced with medium containingAPOPercentage Dye Label. The APOP % Dye Release Reagent was added and amicroplate colorimeter was used to measure cell-bound dye recovered insolution. Apoptotic index was measured at λ-595 nm.

Binding assays were carried out by fluorescence titrations using aFluorolog 2 DMIB spectrofluorometer (SPEX Instruments, Edison, N.J.).FABP5 was bacterially expressed and purified and the equilibriumdissociation constants (Kd) that characterize its interactions with RAand GW0742 were measured by fluorescence competition assays. The methodentails two steps (Lin et al., 1999). In the first step, Kd for theassociation of the protein with the fluorescent fatty acid probe ANS wasmeasured. Protein (1 μM) was titrated with ANS from a concentratedsolution in ethanol. Ligand binding was monitored by following theincrease in the fluorescence of the ligand upon binding to the protein(λ_(ex)-370 nm; λ_(em)-475 nm). Titration curves were analyzed (Norrisand Li, 1998) to yield the number of binding sites and Kd. Kds forbinding of non-fluorescent ligands were then measured by monitoringtheir ability to compete with fluorescent probes for binding. Theprotein was pre-complexed with ANS at 1:1 molar ratio and titrated withRA or GW0742 whose binding was reflected by a decrease in probefluorescence. Kds were extracted from the EC50 of the competition curveand the measured Kd for the probe. Analyses were carried out usingOrigin 7.5 software (MicroCal Software Inc., Northampton, Mass.).

Quantitative real-time PCR (Q-PCR). RNA was extracted and cDNA generatedusing Gene Amp RNA PCR (Applied Biosystems, Foster City, Calif.). Q-PCRanalyses for PDK-1 were conducted using TaqMan chemistry and Assays onDemand probes (Applied Biosystems, PDK1-Hs00176884_m1,ADRP-Hs00765634_m1, FIAF-Hs00211522_m1), Cyp26a-Hs00175671-g1,BTG2-Hs00198887_m1, cyclin D1 0 Hs00277039-m1. 18S ribosomal RNA(4319413E-0312010) was used as a loading control. Analyses were carriedout using the relative standard curve method (Applied BiosystemsTechnical Bulletin no. 2).

Confocal fluorescence microscopy. COS-7 cells were plated in 35 mm glassbottom microwell dishes (Mattek) in DMEM containing 5% charcoal-treatedFBS (75,000 cells per plate), grown for 12 hr. and transfected usingFugene (Roche) with an expression vector harboring GFP-tagged FABP5(EGFP, 250 ng DNA per plate). Following a 48 hr incubation, medium wasreplaced with serum-free DMEM, and live cells were imaged using a LeicaTCS SP2 confocal microscope equipped with a 40× oil immersion lens.After imaging, cells were treated with ligands (1 μM), incubated at 37°C. for 30 min and imaged again. An average of 40 cells were analyzedusing ImageJ (National Institute of Health).

Chromatin immunoprecipitation assays. Nearly confluent HaCat cells weretreated with vehicle or RA (1 μM, 45 min.). Proteins were cross-linkedto DNA (1% formaldehyde, 10 min.). Cells were washed with PBS, scraped,collected, lysed (1% SDS, 10 mM EDTA, 50 mM Tris, pH 7.9, 1 mM DTT, andprotease inhibitors (Roche), and incubated on ice (45 min.). Sampleswere sonicated three times, and chromatin precleared with protein Abeads (2 hr.). Antibodies against PPARβ/δ (H-74, Santa Cruz) orpre-immune rabbit IgG were added and mixtures incubated overnight at 4°C. Protein A beads were added and mixed (2 hr., 4° C.). Beads werewashed twice with low-salt buffer (150 mM NaCl, 0.5% deoxycholate, 0.1%Nonidet P-40, 1 mM EDTA, 50 mM Tris-HCl), twice with high salt buffer(low salt buffer+500 mM NaCl), and twice with Tris-EDTA buffer.Cross-link was then reversed (100 mM NaHCO₃, 1% SDS, overnight 65° C.),proteins digested with proteinase K (1 hr.), and DNA purified(nucleotide extraction kit, Qiagen). The PPRE containing regions of ADRPand FIAF were amplified by PCR using the following primers. ADRP:5′-CCTCTGCTTCACAGGCAAATA-3′ (forward) (SEQ ID NO: 1) and5′-TGCATCAGAAGACTCTCGCCCTTT-3′ (reverse) (SEQ ID NO: 2); FIAF:5′-AATCATGGAAGCCACACTGGTGGT-3′ (forward) (SEQ ID NO: 3) and5′-CCCTACTTTCCTCCCATCCAGTAA-3′ (reverse) (SEQ ID NO: 4). Primersspecific for a region 6-kb upstream of the GAPDH promoter,5′-TCACGCCTGTAATCCCAGCACTTT-3′ (forward) (SEQ ID NO: 5) and,5′-TGATTTCGGCTCACTACAACCTCC-3′ (reverse) (SEQ ID NO: 6), were used as anegative control.

Example 2

One part of our concept is that retinoic acid sensitizes cells toinsulin action, and that it does so by activating the transcriptionfactor PPAR β/δ

To test this hypothesis, we treated cultured adipocytes with: 1) GW0742,a synthetic ligand for PPAR β/δ; 2) TTNPB, a synthetic ligand for theclassical retinoic acid receptor (RAR); and 3) retinoic acid. We thenused real-time PCR to examine the effects of these ligands on theexpression of PDK1 and another known PPAR β/δ target gene, FIAF.

The data in FIG. 4 shows that both FIAF and PDK1 are upregulated inthese cells in response to the PPAR β/δ-ligand, attesting to thepresence and functionality of this receptor in adipocytes. The data alsoshow that, while activation of RAR by TTNPB resulted in down-regulationof the two genes, retinoic acid mimicked the PPAR β/δ-ligand andenhanced the expression of these genes.

Hence, retinoic acid activates PPAR β/δ in adipocytes, thereby inducingthe expression of PDK1, a protein that plays central roles in mediatingthe cell responsiveness to insulin.

These observations suggest that retinoic acid will significantlysensitize cells to insulin actions such as the hallmark of insulinactivity: enabling glucose uptake.

Example 3

We have now completed a pilot experiment to examine the notion thatall-trans-retinoic acid (RA) suppresses obesity and insulin resistance.Experiments were carried out using both cultured adipocytes and adiet-induced mouse model of obesity.

For the in vivo experiments, C57BU6J mice were fed a high fat/highsucrose diet for 16 weeks, when they weighed 1.5 fold more than a cohortfed a standard diet. The obesity of these mice resulted in insulinresistance, the underlying cause for the development of diabetes. Obesemice were separated into two groups. Both groups were continuallymaintained on the high fat/high sucrose diet, and one of them wassystemically treated with RA.

A five week RA treatment led to a loss of ˜15% body weight (FIG. 5)

Weight loss in the mice stemmed almost exclusively from reduced weightof fat tissue, which decreased by over two fold (FIG. 6).

Remarkably, the food consumption of RA-treated mice was higher than thatof the non-treated cohort (FIG. 7). Hence, the reduced adiposity stemmedfrom enhanced energy utilization rather than lower intake.

An important measure of insulin sensitivity is the ability of an animalto clear glucose from plasma. This parameter is assessed by glucosetolerance tests (GTT) in which glucose is administered to the mice andthe kinetics of its clearance from blood measured. As shown in FIG. 8,obese mice displayed a significantly more sluggish response in astandard GTT test as compared with lean mice. Strikingly, RA treatmentimproved glucose tolerance tests to an extent that their response wassimilar to that of lean mice.

One detrimental consequence of insulin resistance is the developmenthepatic steatosis, i.e., accumulation of lipids in liver. As shown inFIG. 9, livers of obese mice displayed a high level of hepatic lipidaccumulation. In contrast, livers of mice fed a high fat diet andtreated with RA were all but devoid of lipid stores.

CONCLUSIONS

In vivo, RA markedly suppresses adiposity and protects against insulinresistance even in the face of a high intake of a high fat/high caloriefood.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. For example, itshould be appreciated that the expression of FABP5 may upregulated incells by, for example gene therapy, to increase insulin sensitivityand/or survival of the cells either alone or in conjunction with theadministration of the all-trans-retinoic acid. Additionally, it will beappreciated that the all-trans-retinoic can be administered with otheragents, such insulin and other insulin sensitizers to treat metabolicdisorders. Such improvements, changes and modifications within the skillof the art are intended to be covered by the appended claims.

1. A method of increasing the insulin sensitivity of insulin resistantcells in a subject with metabolic syndrome, administering to the cellsan amount of all-trans-retinoic acid effective to activate transcriptionfactor perosixome proliferator-activated receptor (PPAR) β/δ of thecells.
 2. The method of claim 1, the all-trans-retinoic acid beingadministered at amount effective to induce expression in the cells of atleast one of 3-phosphoinositide-dependent protein kinase 1 (PDK-1),fasting induced adipose factor (FIAF) or adipose differentiation-relatedprotein (ADRP).
 3. The method of claim 1, the insulin resistant cellscomprising insulin resistant adipocytes.
 4. A method of treatingmetabolic syndrome caused by insulin resistance of cells in the subject,comprising: administering to the subject an amount of all-trans-retinoicacid effective to increase the insulin sensitivity of the insulinresistant cells and treat the metabolic syndrome.